DNA sequencing technology has improved dramatically over recent years with orders of magnitude improvements in the yield of sequence data having resulted from applying massively parallel approaches. Helicos has taken next generation sequencing even further with its single molecule technology, allowing simpler sample prep, lower sample quantities, and lower costs than competing platforms. However, this cost is still not low enough to make whole genome sequencing routinely available for medical or other applications. For sequencing whole genomes to become widespread, it will be necessary to further lower the cost to $1000 or less. With the improvements that can be accomplished via this grant over the next two years, this goal could be realized. Sequencing of a whole human genome using single-molecule sequencing with a HeliScope has already been accomplished for less than $100,000 outside of a genome center and technical improvements to that system should make it possible to bring the price down to less than $1000. During the span of our current grant (now in the final year of NIH grant 5R01HG004144, High Accuracy Single Molecule DNA Sequencing by Synthesis), Helicos has been able to surmount all the critical technical barriers for single-molecule sequencing at the genome-wide scale. We have taken a very early stage technology and driven it to a commercial instrument which, even in its initial state, provides sequence data at an unprecedented low cost. The next two years are critical for affirming the commercial viability of this platform and technology. Attainment of additional improvements in yield, read length, and error rates based on improvements to the sequencing surface and better nucleotides would allow us to drive costs into the <$1000 range for a whole human genome. By developing an ordered surface of DNA primers, up to 5x increase in sequencing yield is possible. Similarly, dyes with better fluorescence yield will allow reduced imaging time and higher throughput. Additionally, many other biological applications that are not possible to carry out with other technologies will be enabled with these improvements. Because many potential improvements are additive, it is not necessary to make substantial gains in all areas but, rather, measured improvements in the proposed areas would be sufficient. As such, we aim to attack the issues of cost and throughput on multiple fronts in order to improve all aspects of the process and allow attainment of the long sought $1000 genome. PUBLIC HEALTH RELEVANCE: Massively parallel DNA sequencing has revolutionized many areas of biology by providing orders of magnitude more sequence data than previously possible. The majority of this sequence data has been generated using systems that require complex sample preparation methods, which limits the applications that can be attempted and increases the cost. By improving the current true single-molecule sequencing system to lower costs and improve yield, additional applications will become possible. These applications will likely include basic research into disease pathogenesis, as well as clinical and applied research. Therefore, the proposed project will open new research avenues, lead to a better understanding of the biological mechanisms underlying disease states, and ultimately aid in identifying revolutionary new ways to diagnose, treat and prevent human disease.